The overall goal of this project is to investigate strategies for the drug treatment of epilepsy through pharmacological studies in animal models and clinical investigation in human subjects. Research was continued evaluating the role of neuroactive steroids in epilepsy and their possible uses in epilepsy therapy. Neuroactive steroids are endogenous steroid hormones (and their synthetic analogs) that rapidly alter the excitability of neurons by direct actions on membrane ion channels, including GABA-A and NMDA receptors. In prior reporting periods, we confirmed that the reproductive hormone progesterone has powerful anticonvulsant activity and we demonstrated that this results from its conversion to the neuroactive steroid allopregnanolone. We proposed that perimenstrual catamenial epilepsy, the increase in seizure frequency that many women with epilepsy experience near the time of menstruation (when progesterone levels fall) may, in part, be related to withdrawal of allopregnanolone. At present, there is no specific treatment for catamenial epilepsy. However, our studies with an animal model of catamenial epilepsy suggested that neurosteroid replacement could be useful. We have initiated clinical studies to validate the neurosteroid withdrawal hypothesis of catamenial epilepsy and we plan a clinical trial to evaluate the utility of neurosteroid replacement. In the present reporting period we completed a study examining the role of neurosteroids in stress-induced alterations in seizure susceptibility, focusing specifically on tetrahydrodeoxycorticosterone (DOC), an adrenal steroid whose synthesis is enhanced during stress. DOC is conventionally considered a mineralocorticoid precursor; however, we obtained evidence indicating that it is also a mediator of stress effects on seizures. DOC undergoes sequential metabolic reduction by 5alpha-reductase and 3alpha-hydroxysteroid oxidoreductase to form dihydrodeoxycorticosterone (DHDOC) and THDOC, which is a GABA-A receptor modulating neurosteroid with anticonvulsant properties. Acute swim stress in rats significantly elevated plasma THDOC concentrations and raised the PTZ seizure threshold. Small systemic doses of DOC produced comparable increases in plasma THDOC levels and PTZ seizure threshold. Pretreatment with finasteride, which blocks the conversion of DOC to DHDOC, reversed the antiseizure effects of stress. DOC also protected mice against PTZ, DMCM (methyl-6,7-dimethoxy-4-ethyl-beta-carboline-3-caboxylate), picrotoxin and amygdala kindled seizures in mice. Finasteride reversed the antiseizure activity of DOC; partial antagonism was also obtained with indomethacin, an inhibitor of 3alpha-hydroxysteroid oxidoreductase. Finasteride had no effect on seizure protection by DHDOC and THDOC whereas indomethacin partially reversed DHDOC but not THDOC. The results with finasteride and indomethacin indicated that THDOC and possibly DHDOC could mediate the anticonvulsant activity of DOC. In whole cell voltage clamp recordings from cultured hippocampal neurons, we found that DHDOC, like THDOC, potentiated GABA-activated chloride currents and directly activated GABA-A receptor currents compatible with a role for DHDOC in the antiseizure activity of DOC. Our results demonstrate that DOC is a mediator of the physiological effects of acute stress that could contribute to stress-induced changes in seizure susceptibility through its conversion to neurosteroids with modulatory actions on GABA-A receptors including THDOC and possibly also DHDOC. Our results further suggest a role for neuroactive steroids as a treatment approach for stress-related seizures. In the present reporting period we also carried out studies examining the role of the progesterone receptor (PR), a nuclear transcription factor, in mediating the anticonvulsant effects of progesterone. Although our prior research demonstrated that progesterone modulation of seizure susceptibility occurs indirectly through its neurosteroid metabolite allopregnanolone, since the PR is the primary target of progesterone action in diverse tissues, we sought to exclude the possibility that progesterone's effects on seizures in part occur through transcriptional effects mediated by the PR. In these studies, we used PR """"""""knockout"""""""" (PRKO) mice in which the PR gene had been deleted by gene targeting (resulting in the absence of both the A and B forms of the PR). Progesterone protected PRKO mice against pentylenetetrazol (PTZ) and amygdala-kindled seizures, and the antiseizure activity of progesterone was reversed by pretreatment with finasteride. The endogenous neurosteroids allopregnanolone and THDOC, and the synthetic neuroactive steroid ganaxolone also exhibited potent anticonvulsant activity in PRKO mice. However, we unexpectedly found a 2-fold increase in the antiseizure potency of progesterone in PRKO mice compared to wild type controls. In addition, we observed that PRKO mice have an overall reduced susceptibility to PTZ seizures. To begin to understand the molecular basis for these alterations in seizure susceptibility, we have carried out gene expression analysis using a microarray representing 12,000 sequences including all 6,000 functionally characterized sequences in the mouse UniGene database. We hypothesized that the effects of deletion of the PR could be due to alterations in the expression of GABA-A receptor subunit genes. However, a comprehensive analysis of all known subunits revealed no significant differences between PRKO and wild-type animals. We also surveyed all known ionotropic glutamate receptor genes. We found that only KA2 (high affinity kainate receptor subunit that forms heteromeric channels with GluR5 and GluR6) and NMDAR1 (obligatory subunit of NMDA receptors) showed significant differential expression, with an increase in PRKO animals of about two-fold in both cases. In addition, the microarray analysis showed twenty other genes whose expression was increased and two that were decreased. The change in KA2 subunit expression was of particular interest inasmuch as heteromeric kainate receptors show more rapid desensitization as compared with homomeric receptors and increased abundance of KA2-containing kainate receptors could account for the reduced propensity for seizures. As a complement to our prior studies on drug and hormonal treatment of epilepsy, we have begun a new research direction to investigate alternative epilepsy treatment approaches. Our current emphasis is on the use of convection-enhanced brain infusion for the suppression or ablation of epileptic foci. We completed a study in collaboration with the Surgical Neurology Branch examining the long-term antiseizure effects of infusion of the excitotoxin ibotenate in a rat model of temporal lobe epilepsy. Kainate injection into the left amygdala of rats elicited chronic spontaneous limbic seizure activity. Two weeks after the injection, ibotenate, a nonepileptogenic excitatory amino acid that is an axon-sparing neuronal cell toxin, was infused into the left amygdala and piriform lobe. The infusion eliminated the electrical and behavioral seizure activity. We conclude that lesioning of an epileptic focus by convective distribution of ibotenate can produce an enduring suppression of seizure activity. These studies demonstrate a potential chemical neurosurgical approach to epilepsy therapy that could represent an alternative to conventional ablative neurosurgical procedures in the management of partial epilepsies.
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